This study will focus on factors that allow the obligate anaerobe B.fragilis, although a component of the normal colonic microbiota, to be a successful pathogen. These include its ability to withstand an aerobic environment (aero-tolerance) during early stages of infection; the presence of systems to import heme into the cell for the heme-dependent pathways of central metabolism and defense against reactive oxygen species; the ability of B.fragilis to remove sialic acid residues from host components, and its virtuosity in obtaining nutrients for growth in vivo from complex oligosaccharides and glycoproteins. Specific aims include: 1, to continue to study factors that allow B.fragilis to withstand prolonged oxygen challenge (aerotolerance): We propose that activities in the B.fragilis periplasm serve as the initial line of defense to combat the formation of reactive oxygen species (ROS), protect sensitive targets from ROS challenges and to reverse ROS damage. In addition we have identified specific functions (superoxide dismutase, SOD), and an extensive gene cluster (the Bat operon) that are required for aerotolerance. We will test the hypothesis that the Bat operon plays an important role in exporting reducing potential from the cytoplasm to the periplasm. 2. Acquisition of iron and heme is important for B.fragilis growth in vitro and in vivo. We will study the process of heme uptake in B.fragilis by the heme permease systems whose genes and functions we have described. We will also continue to study the heme-dependent, and Fe-S cluster-containing enzymes in the dual pathways of central metabolism to establish their roles in aerotolerance and in providing energy during oxygen challenge. 3. to investigate the composition, functions and control of operons for the acquisition of growth substrates from the infected host. We will focus on the operon containing the neuraminidase (nanH1) gene and several other glycohydrolases capable of converting the complex Lewis antigen found on the surface of many human cells to individual monosaccharides. We will continue to define the operon for NANA utilization, the NanL1 operon. Is neuraminidase a virulence factor because it supplies NANA for B.fragilis growth, or because its activity alters the surface of host cells during infection, or both? 4.We will develop additional tools to define changes in macromolecular synthesis and stability of DNA, proteins and membranes during 9 oxygen challenge.